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TRRAP-mediated regulation of Wee1

ABSTRACT: The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Up-regulation of Chk1 by mutation or overexpression bypasses the requirement for up-stream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed Chk1. We identified a mutation in tra1, which encodes one of two homologs of TRRAP, an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed Chk1, suggesting this phenotype is due to a HAT activity, though expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing Chk1, and accumulate inactive Wee1 protein. The Cdr (changed division response) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and while best characterized in the cellular response to limited nutrition, we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE12674 | GEO | 2010/04/11

SECONDARY ACCESSION(S): PRJNA112747

REPOSITORIES: GEO

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TRRAP-mediated regulation of Wee1

Project description:The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Up-regulation of Chk1 by mutation or overexpression bypasses the requirement for up-stream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed Chk1. We identified a mutation in tra1, which encodes one of two homologs of TRRAP, an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed Chk1, suggesting this phenotype is due to a HAT activity, though expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing Chk1, and accumulate inactive Wee1 protein. The Cdr (changed division response) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and while best characterized in the cellular response to limited nutrition, we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation. Two and three independent microaaray experiments were done for wild type and the mutant (tra1-1) respectively.

2010-04-11 | E-GEOD-12674 | biostudies-arrayexpress

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress

Project description:While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A confers cellular resistance to CHK1 inhibitors and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase which dephosphorylates the WEE1 protein and rescues WEE1 from Ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1 inhibitors. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1 inhibitor plus a WEE1 inhibitor can overcome CHK1 inhibitor resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1 inhibitor sensitivity or resistance.

2021-01-01 | GSE158338 | GEO

Development and Characterization of a Wee1 Kinase Degrader

Project description:The G1/S cell-cycle checkpoint is frequently dysregulated in cancer, leaving cancer cells particularly reliant on a functional G2/M checkpoint to prevent excessive DNA damage. Inhibiting regulators of the G2/M checkpoint can therefore drive cancer cells into premature mitosis, ultimately causing cell death by mitotic catastrophe. One such regulator is Wee1, a nuclear tyrosine kinase that delays mitotic entry by phosphorylating CDK1 at Tyr15. Previous drug development efforts targeting Wee1 resulted in the clinical-grade inhibitor, AZD1775. However, AZD1775 is burdened by dose- limiting adverse events, and has off-target PLK1 activity. In an attempt to overcome these limitations, we designed small molecule degraders of Wee1 by conjugating AZD1775 to the cereblon (CRBN)- binding ligand, pomalidomide, as informed by molecular docking. The resulting lead compound, ZNL- 02-096, degrades Wee1 while sparing PLK1, induces G2/M phase accumulation at up to 10-fold lower concentrations than AZD1775, and potently synergizes with Olaparib in ovarian cancer cell lines. We demonstrate that ZNL-02-096 has CRBN-dependent pharmacology that is distinct from the conventional catalytic inhibitor, AZD1775, which justifies further evaluation of selective Wee1 degraders.

2019-11-18 | PXD015513 | Pride

Aguda1999 - G2 DNA damage checkpoint

Project description:Baltazar D. Aguda. A quantitative analysis of the kinetics of the G(2) DNA damage checkpoint system. Proceedings of the National Academy of Sciences 96, 20 (1999). A detailed model of the G(2) DNA damage checkpoint (G2DDC) system is presented that includes complex regulatory networks of the mitotic kinase Cdc2, phosphatase Cdc25, Wee1 kinase, and damage signal transduction pathways involving Chk1 and p53. Assumptions on the kinetic equations of the G2DDC are made, and computer simulations are carried out to demonstrate how the various subsystems operate to delay or arrest cell cycle progression. The detailed model could be used to explain various experiments relevant to G2DDC reported recently, including the nuclear export of 14-3-3-bound Cdc25, the down-regulation of cyclin B1 expression by p53, the effect of Chk1 and p53 on Cdc25 levels, and Wee1 degradation. It also is shown that, under certain conditions, p53 is necessary to sustain a G(2) arrest.

2024-09-02 | BIOMD0000000704 | BioModels

GCN2 is a determinant of the response to WEE1 kinase inhibition in small-cell lung cancer

Project description:Patients with small-cell lung cancer (SCLC) are in dire need of more effective therapeutic options. Frequent disruption of the G1 checkpoint in SCLC cells creates a greater dependency of these cells on the G2/M checkpoint to maintain genomic integrity. Indeed, in pre-clinical models, inhibiting the G2/M kinase WEE1 shows promise in inhibiting SCLC growth. However, toxicity and acquired resistance limit the clinical effectiveness of this strategy. Here we conducted CRISPR/Cas9 knockout screens to identify genes influencing the response of SCLC cells to WEE1 kinase inhibition. These screens uncovered a role for the GCN2 amino acid-sensing pathway in modulating the response of SCLC cells to WEE1 inhibition. Rapid activation of GCN2 upon WEE1 inhibition triggers a stress response. Pharmacological activation of the GCN2 pathway synergizes with WEE1 inhibition. Thus, activation of the GCN2 amino acid-sensing pathway represents a novel approach for augmenting the efficacy of WEE1 inhibitors in SCLC.

2024-07-16 | GSE246354 | GEO

Replication origin mapping of cdc25-22 and Cdc13-Cdc2 cells after G2 block

Project description:Identification of sites of replication initiation by copy number analysis, comparing samples before and after entry into S phase. Experiments were performed in the following strains and conditions: cdc25-22 (temperature-sensitive mutation arrests cells at G2/M for synchrony, otherwise wild type), G2 arrest; Cdc13-Cdc2, G2 arrest.

2018-02-04 | GSE88711 | GEO

Transcriptome analysis of mouse preimplantation embryos when DNA damage checkpoint is perturbed

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2021-05-04 | GSE163765 | GEO

CDK2-mediated TNFa Upregulation as a Mechanism of Cytotoxicity in Acute Leukemia

Project description:Although inhibitors of the kinases CHK1, ATR and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines, including TP53 null acute myeloid leukemia (AML) and BCR/ABL-positive acute lymphoid leukemia (ALL), in vitro and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, CHK1 downregulation or CHK1 inhibitor treatment triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNF production and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically CHK1 inhibitor resistant exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2  AP1  TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to antileukemic cell action of CHK1, ATR and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand-induced killing that can potentially be exploited for acute leukemia treatment.

2020-12-28 | GSE131912 | GEO

Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

Project description:Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells upregulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdc2. In this study we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A but leads to relief of negative feedback inhibition and activation of Wee1-Cdc2 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effectors, like PP2A and Wee1, may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors Biological triplicate of PC3 treated with siClusterin were compared to biological trplicate of PC3 treated with siScramblein

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